Susceptibilities to amphotericin B and fluconazole of Candida species in Taiwan Surveillance of Antimicrobial Resistance of Yeasts 2006

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1 Available online at Diagnostic Microbiology and Infectious Disease 61 (2008) Mycology Susceptibilities to amphotericin B and fluconazole of Candida species in Taiwan Surveillance of Antimicrobial Resistance of Yeasts 2006 Yun-Liang Yang a, An-Huei Wang b, Chih-Wei Wang b, Wei-Ting Cheng b, Shu-Ying Li c, Hsiu-Jung Lo b, TSARY Hospitals a Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 300, Taiwan, ROC b Division of Clinical Research, National Health Research Institutes, Zhunan Town, Miaoli County 350, Taiwan, ROC c Division of Laboratory Research and Development, Center for Disease Control, Taipei 115, Taiwan, ROC Received 27 October 2007; accepted 16 January 2008 Abstract Susceptibilities to amphotericin B and fluconazole of 964 Candida isolates collected in Taiwan Surveillance of Antimicrobial Resistance of Yeasts in 2006 were determined. There were 419 (43.5%) Candida albicans, 246 (25.5%) Candida tropicalis, 211 (21.9%) Candida glabrata, 62 (6.4%) Candida parapsilosis, 14 (1.5%) Candida krusei, and 12 (1.2%) others. Interestingly, 16 of the 17 amphotericin B-resistant isolates were non-albicans Candida species. The resistant rate to amphotericin B has decreased from 2.5% in 2002 to 1.8% in On the other hand, there were 132 C. tropicalis, 14 C. krusei, 10 C. albicans, and 9 C. glabrata isolates that had MICs to fluconazole 64 μg/ml. The prevalence of isolates with such high MICs was significantly higher than that in 2002 (17.1% versus 1.9%). Our results further indicate that most of the isolates with MICs to fluconazole 64 μg/ml exhibited the trailing phenomenon Elsevier Inc. All rights reserved. Keywords: Candida; Susceptibility; Resistance 1. Introduction Because of alterations in immune status and invasive hospital procedures (White et al., 1998; Yang and Lo, 2001), infections caused by opportunistic pathogens, such as yeasts, are becoming important causes of morbidity and mortality in immunocompromised patients. In the past 2 decades, nosocomial yeast infections have increased significantly worldwide. For example, the prevalence of nosocomial candidemia increased 27-fold from 1981 through 1993 at a major hospital in Taiwan (Chen et al., 1997; Hung et al., 1996). In the United States, yeast infection also ranks as the 4th most common cause of nosocomial bloodstream infection (Wisplinghoff et al., 2004). Several antifungal drugs have been applied to render the situation, and as a Corresponding author. Tel.: x35516; fax: address: hjlo@nhri.org.tw (H.-J. Lo). result of broad prophylactic usages and long-term treatments with those drugs, the prevalence of drug resistance has become an important issue in various yeast infections, which have profound effects on human health (Marr et al., 2001; Pfaller et al., 2003; Yang et al., 2004b). Candida species have various degrees of susceptibility to frequently used antifungal drugs. For instance, Candida lusitaniae is relatively resistant to amphotericin B (Hadfield et al., 1987). Candida krusei is intrinsically resistant to fluconazole, and Candida glabrata is less susceptible or has a higher MICs to it than other Candida species (Akova et al., 1991; Orozco et al., 1998; Yang et al., 2004b). This phenomenon illustrates the importance of identification and surveillance of Candida species in the clinical settings. In 1999 and again in 2002, 2 national surveys in Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) have been conducted. The drug susceptibilities of the 632 and 909 isolates collected in 1999 and 2002, respectively, have been determined (Yang et al., 2004b, 2005b). Among them, 0.5% (1999) and 2.5% (2002) were resistant to /$ see front matter 2008 Elsevier Inc. All rights reserved. doi: /j.diagmicrobio

2 176 Y.-L. Yang et al. / Diagnostic Microbiology and Infectious Disease 61 (2008) amphotericin B. There were 8.4% of the isolates in 1999 that had MICs to fluconazole 64 μg/ml, whereas only 1.9% in 2002 (Yang et al., 2004b, 2005b). In 2006, a follow-up survey was taken place, and 964 Candida isolates were collected and analyzed. The article is to report the result of the susceptibilities to antifungal drugs of Candida species in TSARY 2006 and the trends of the resistance in Taiwan from 1999 to Materials and methods 2.1. Organisms and media Yeast isolates were collected according to previous studies (Lo et al., 2001; Yang et al., 2005b) from the 22 hospitals participating in TSARY from July to September in Each hospital was asked to submit all yeast pathogens from blood and the first 10 Candida albicans and 40 non-albicans Candida species isolates from nonsterile sites. In principle, only 1 isolate was accepted from each specimen. Nevertheless, when there were multiple species isolated from 1 specimen, 1 isolate from each species was analyzed. All the collected isolates were stored frozen at 70 C in bead-containing Microbank cryovials (Pro- Lab Diagnostics, Austin, TX). After their arrival at the laboratory at National Health Research Institutes (NHRI), Taiwan, ROC, these isolates were 1st subcultured on Sabouraud dextrose agar (Becton Dickinson, Cockeysville, MD) to assess the purity and identification. Pure isolates were labeled and stored in vials containing 50% glycerol at 70 C awaiting further analyses Identification The identifications of the isolates were reassessed in the laboratory at the NHRI. The identification procedure for the yeast isolates was modified based on our previous report (Lo et al., 2001). All isolates were subjected to API-32C (biomérieux, St. Louis, MO). The VITEK Yeast Biochemical Card (YBC, biomérieux) would then be used for the identification of the isolates when the API-32C showed less than 90% confidence and for the isolates whose information was inconsistent to that provided by the hospitals. The sequence of internal transcribed spaces of ribosomal DNA (Leaw et al., 2006, 2007) was applied for further assessment when both API-32C and YBC failed Antifungal susceptibility testing The MICs to amphotericin B and to fluconazole of each isolate were determined by the same in vitro antifungal susceptibility testing as those in TSARY 1999 and TSARY 2002 (Yang et al., 2004b, 2005b), according to the guidelines of M27A published in 1997 by the Clinical and Laboratory Standards Institute (CLSI, 1997). The RPMI medium 1640 ( ) provided by Gibco BRL was used for the testing. Strains from American Type Culture Collection including C. albicans (ATCC 90028), C. krusei (ATCC 6258), and Candida parapsilosis (ATCC 22019) were used as the standard controls. The final growth of each isolate was measured by Biotrak II plate reader (Amersham Biosciences, Cambridge, UK) after incubation at 35 C for 48 h. The MICs of some isolates were also measured by Etest (AB BIODISK, Solna, Sweden) to assess the results of the broth microdilution method. The MICs to amphotericin B and to fluconazole were defined as the MICs of drugs capable of reducing the turbidity of cells to greater than 95% and 50%, respectively. For susceptibility to amphotericin B, isolates with MIC 2 μg/ml were considered to be resistant, and those with MIC 1 μg/ml were susceptible. For susceptibility to fluconazole, isolates with MIC 64 μg/ml were considered to be resistant, whereas those with MIC 8 μg/ml were susceptible. Isolates with MICs falling in between (16 32 μg/ml) were susceptible dose dependent. The MICs of 50% and 90% of the total population were defined as MIC 50 and MIC 90, respectively. Among the phenomena associated with resistance, trailing describes the reduced but persistent growth that some isolates exhibit at drug concentrations above the MIC in broth dilution tests with azole antifungal agents, such as fluconazole (Lee et al., 2004). When the MIC of an isolate measured after 48-h incubation is approximately 4-fold higher than that at the 24-h point (Arthington-Skaggs et al., 2002), the isolate is defined to have trailing growth Database and analysis The database for this study contained the characteristic information of each submitted isolate: hospital origin, location and type of the hospital, and identification and source of the isolate. The statistic significance of the differences in frequencies and proportions was determined by the χ 2 test with Yates' correction. 3. Results and discussion 3.1. Distribution of Candida species The distribution of Candida species in TSARY 2006 (Table 1) was similar to that of 2 previous surveys in 1999 and 2002 (Yang et al., 2004b, 2005a). C. albicans was the most frequently isolated species, accounting for 43.5% of the total isolates. Candida tropicalis (25.5%) and C. glabrata (21.9%) were the 2 most frequently isolated non-albicans Candida species, followed by C. parapsilosis (6.4%), C. krusei (1.5%), and others (1.2%). When classified according to the sources (Table 1), there were 406 (42.1%) isolates from urine, 158 (16.4%) from sputum, 145 (15%) from blood, 43 (4.5%) from wound, 40 (4.1%) from pus, 40 (4.1%) from catheter tip, 38 (4%) from ascites, and 94 (9.8%) from other 45 different sites. Different Candida species had various prevalence in different body sites. Although C. albicans was still the most

3 Y.-L. Yang et al. / Diagnostic Microbiology and Infectious Disease 61 (2008) Table 1 The distribution of Candida species from different sources Sources C. albicans C. tropicalis C. glabrata C. parapsilosis C. krusei Others Total Urine (42.1) a Sputum (16.4) Blood (15.1) Wound (4.5) Pus (4.1) Tip b (4.1) Ascites c (3.9) Others (9.8) Total 419 (43.5) 246 (25.5) 211 (21.9) 62 (6.4) 14 (1.5) 12 (1.2) 964 a Number of isolates (percentage). b Catheter tips. c Body fluid, not urinary ascites or pus. common single species (56.6%) causing candidemia in our study as well as others' (Cheng et al., 2004; Peman et al., 2005; Pfaller et al., 2007a; Yang et al., 2006), the prevalence of non-albicans Candida species had increased. Though C. tropicalis was the most common non-albicans Candida species in our collection, C. glabrata was the most common one from urine (145/277, 52.3%). The majority of C. glabrata (68.7%) was isolated from urine samples, consistent with the previous reports that C. glabrata is 2nd only to C. albicans as a cause of candiduria (Pfaller et al., 1999; Yang et al., 2004b) Susceptibilities to amphotericin B of Candida species The range of MICs to amphotericin B of the 964 isolates was from to 2 μg/ml (Table 2). C. krusei was less susceptible to amphotericin B than any other species because the MIC 50 of this species was 1 μg/ml. The overall resistant rate to amphotericin B has decreased from 2.5% in TSARY 2002 (Yang et al., 2005b) to 1.8% in TSARY In recent years, fungal infections caused by non-albicans Candida species have increased dramatically (Abi-Said et al., 1997; Slavin et al., 1995; Walsh et al., 2004). The phenomenon was also reflected in the distribution of resistant isolates in others' (Slavin et al., 1995; Walsh et al., 1998) as well as our studies. All 3 amphotericin B-resistant isolates in TSARY 1999 were non-albicans Candida species (Yang et al., 2004b). Furthermore, 20 of the 23 amphotericin B-resistant isolates in TSARY 2002 were also non-albicans Candida species (Yang et al., 2005b). Of the 17 amphotericin B-resistant isolates in TSARY 2006, 16 were non-albicans Candida species. They consisted of 12 C. tropicalis, 2 C. krusei, and 1 each of C. glabrata and Candida curvatus. The prevalence of amphotericin B resistance of C. krusei (14.3%) or C. tropicalis (4.9%) was significantly higher than that of C. glabrata (0.5%) or C. albicans (0.2%) (P b 0.05). Among the frequently isolated non-albicans Candida species, no amphotericin B-resistant isolate of C. parapsilosis was detected in this study (Table 2), as well as in the 2 previous TSARYs (Yang et al., 2004b, 2005b). A total of 2, 2, and 5 C. lusitaniae were collected in TSARY 1999, 2002, and 2006, respectively. Up-to-date amphotericin B resistance of C. lusitaniae is not an issue for concern since all 9 C. lusitaniae was susceptible to amphotericin B. The MIC 50 and MIC 90 of those isolates were 0.25 and 1 μg/ml, respectively Susceptibilities to fluconazole of Candida species from different sources For the susceptibility to fluconazole, a total of 756 (78.4%), 43 (4.5%), and 165 (17.1%) isolates had MICs 8, 16 to 32, and 64 μg/ml, respectively. The MIC 50 and MIC 90 of these isolates were 1 and 64 μg/ml, respectively. Interestingly, among the isolates with MICs 64 μg/ml, those from sputum (27.2%) appear to have a higher ratio than Table 2 The susceptibility to amphotericin B of Candida species MICs (μg/ml) C. albicans C. tropicalis C. glabrata C. parapsilosis C. krusei Others Total (3.8) a 0 4 (1.9) (8.3) 21 (2.2) (50.8) 45 (18.3) 49 (23.2) 23 (37.1) 0 6 (50) 336 (34.8) (31.9) 126 (51.2) 113 (53.6) 29 (46.8) 5 (35.7) 2 (16.7) 409 (42.4) 1 55 (13.1) 63 (25.6) 44 (20.8) 10 (16.1) 7 (50) 2 (16.7) 181 (18.8) 2 1 (0.2) 12 (4.9) 1 (0.5) 0 2 (14.3) 1 (8.3) 17 (1.8) Total MIC MIC a Number of isolates (percentage).

4 178 Y.-L. Yang et al. / Diagnostic Microbiology and Infectious Disease 61 (2008) those from ascites (7.9%), wound (11.6%), blood (13.1%), and urine (17.7%) (P = 0.05) (Table 3). When comparisons were made among species, there were distinct variations in the fluconazole susceptibility. Of the 5 common Candida species, all C. krusei had MICs 64 μg/ ml. In contrast, none of the C. parapsilosis did. These results were consistent with previous reports (Akova et al., 1991; Pfaller et al., 2000, 2004; Yang et al., 2004a, 2005b). Furthermore, susceptibilities among different species from blood also varied. All C. albicans, C. glabrata, and C. parapsilosis were susceptible to fluconazole, whereas 18 of the 32 (56.3%) C. tropicalis isolates had MICs 64 μg/ml (Table 3). Interestingly, C. glabrata having MICs to fluconazole 64 μg/ml was lower than what has been reported in other geologic areas (Fleck et al., 2007; Pfaller et al., 2007b; St Germain et al., 2001). In the 3 TSARYs, the portions of isolates having MICs 64 μg/ml were 8.3%, 1.6%, and 4.3% isolates from 1999, 2002, and 2006, respectively. It has been reported that continuous exposure to azoles seems to have a major impact on developing resistance to fluconazole in Candida species, especially for C. glabrata (Kontoyiannis, 2002). Therefore, our previous observation that only 13% of the candidemia 92 patients had prior fluconazole treatments (Cheng et al., 2004) may explain the low fluconazole-resistant C. glabrata in Taiwan Trailing growth Among the phenomena associated with resistance, trailing describes the reduced but persistent growth that some isolates exhibit at drug concentrations above the MIC in broth dilution tests with azole antifungal agents, such as fluconazole (Lee et al., 2004). Trailing may interfere the observation of resistance level in vivo (Arthington-Skaggs et al., 2002). Thus, we have also determined whether those 165 isolates with MICs 64 μg/ml to fluconazole exhibit Table 3 The susceptibility to fluconazole of Candida species from different sources MICs (μg/ml) C. albicans C. tropicalis C. glabrata C. parapsilosis C. krusei Others Total Urine (95.3) a 53 (48.6) 117 (80.7) 13 (100) 0 5 (100) 311 (76.6) (0.9) 22 (15.2) (5.7) 64 6 (4.7) 55 (50.5) 6 (4.1) 0 5 (100) 0 72 (17.7) Subtotal Sputum 8 69 (98.6) 25 (38.5) 13 (68.4) 2 (100) (69) (1.4) 0 5 (26.3) (3.8) (61.5) 1 (5.3) 0 2 (100) 0 43 (27.2) Subtotal Blood 8 82 (100) 10 (31.2) 16 (80) 9 (100) 0 1 (100) 118 (81.4) (12.5) 4 (20) (5.5) (56.3) (100) 0 19 (13.1) Subtotal Wound 8 21 (100) 2 (3.3) 5 (83.3) 5 (100) 0 3 (75) 36 (83.7) (16.7) (25) 2 (4.7) (66.7) (100) 0 5 (11.6) Subtotal Pus 8 19 (95) 4 (44.4) 2 (100) 6 (100) 0 1 (100) 32 (80) (5) 5 (55.6) (100) 0 8 (20) Subtotal Tip b 8 18 (90) 7 (53.8) 2 (66.7) 4 (100) (77.5) (7.7) (2.5) 64 2 (10) 5 (38.5) 1 (33.3) (20) Subtotal Ascites c 8 24 (100) 5 (71.4) 4 (100) 2 (100) (92.1) (28.6) (100) 0 3 (7.9) Subtotal Others 8 51 (96.2) 2 (40) 10 (83.4) 20 (95.2) (100) 84 (89.4) (1.9) (8.3) 1 (4.8) (3.2) 64 1 (1.9) 3 (30) 1 (8.3) 0 2 (100) 0 7 (7.4) Subtotal All (97.1) 108 (43.9) 169 (80.1) 61 (98.4) 0 11 (91.7) 756 (78.4) (0.5) 6 (2.4) 33 (15.6) 1 (1.6) 0 1 (8.3) 43 (4.5) (2.4) 132 (53.7) 9 (4.3) 0 14 (100) (17.1) Total a Number of isolates (percentage). b Catheter tips. c Body fluid, not urinary ascites or pus.

5 Y.-L. Yang et al. / Diagnostic Microbiology and Infectious Disease 61 (2008) Table 4 The distribution of trailing growth of Candida species with MICs greater than 64 μg/ml Sources C. albicans trailing, and the results are summarized in Table 4. High percentage (64.8%) of those isolates indeed exhibited the trailing phenomenon. When classified according to species, 72.7% (96/132) C. tropicalis, 70% (7/10) C. albicans, and 44.4% (4/9) C. glabrata isolates exhibited the trailing phenomenon, whereas none of the C. krusei isolates did Conclusion C. glabrata C. krusei C. tropicalis Subtotal Total No Yes No Yes No Yes No Yes No Yes Urine Sputum Blood Pus Ascites Wound Tip Others Total In TSARY 1999, there were 14.7% C. tropicalis isolates having MICs 64 μg/ml (Yang et al., 2004b). In contrast, none of the 244 C. tropicalis from TSARY 2002 had MICs 64 μg/ml (Yang et al., 2005b). Surprisingly, in TSARY 2006, among the 246 C. tropicalis isolates, 132 (53.7%) had MICs 64 μg/ml. Recently, we have reported an association between fluconazole susceptibility and genetic relatedness among C. tropicalis isolates from TSARY 1999 (Chou et al., 2007; Wang et al., 2007). The DST140 was a predominant type of C. tropicalis isolates among those having MICs 64 μg/ml in TSARY 1999 (Chou et al., 2007). Furthermore, none of the 17 tested fluconazolesusceptible C. tropicalis isolates collected from TSARY 2002 were DST140 type (Chou et al., 2007). Interestingly, our preliminary result showed that DST140 was detected again among C. tropicalis isolates collected in TSARY This result may explain the dramatic fluctuation in fluconazole susceptibility among 3 TSARYs. Whether this is an endemic problem requires further investigation. The increasing rate of reduced susceptibility to fluconazole in C. tropicalis has considerable clinical importance, because this species is 1 of the most frequently isolated nonalbicans Candida species (Cheng et al., 2004; Hung et al., 2005; Lo et al., 2005; Pfaller et al., 2000; Prasad et al., 1999). Furthermore, C. tropicalis develops drug resistance in the presence of fluconazole much more rapidly than C. albicans (Barchiesi et al., 2000; Calvet et al., 1997). In addition, C. krusei isolates with high MICs are also closely related (Chou et al., 2007; Wang et al., 2007). These findings may explain why, to fluconazole, C. tropicalis has a higher rate to have MICs 64 μg/ml than C. albicans. Here we have identified that 72.7% (96/132) C. tropicalis exhibited the trailing phenomenon by a definition that the MIC of an isolate after 48-h incubation is 4-fold higher than that at the 24-h point. Nevertheless, whether the remaining 36 (14.6%) C. tropicalis isolates are truly resistant requires further investigation. Acknowledgments The authors would like to thank Bristol Myers Squibb (New Brunswick, NJ) and Pfizer (New York, NY) for supplying the amphotericin B and fluconazole, respectively. They would also like to acknowledge Dr. Y.C. Chen for her assistance and helpful suggestion. They also thank the 22 participating hospitals for providing clinical isolates and information regarding those isolates. They are Asia East Memorial Hospital, Buddhist Tzu-Chi General Hospital, Chang Gung Memorial Chiayi Christian Hospital, Hospital at Kaohsiung, Chang-Hwa Christian Hospital, Cheng Ching Hospital, Chung Shan Medical Dental College Hospital, Kaohsiung Military Hospital, Kaohsiung Medical College Chung-Ho Memorial Hospital, Kuan-Tien General Hospital, Lo-Hsu Foundation Lo-Tung Poh Ai Hospital, Miin Sheng General Hospital, National Cheng Kung University Hospital, Show Chwan Memorial Hospital, Sin-Lau Christian Hospital, St. Mary Hospital, Taipei Municipal Chung Hsiao Hospital, Taipei Municipal Hoping Hospital, Veterans General Hospital-Taichung, Veterans General Hospital-Kaohsiung, Zen Ai General Hospital, and Tungs' Taichung NetroHarbor Hospital, Sha Lu branch. We thank Dr. T.L. Lauderdale and Mr. S.H. Hsu for their technical assistance. This study was supported by the grant NHRI CL-096-PP07. References Abi-Said D, Anaissie E, Uzun O, Raad I, Pinzcowski H, Vartivarian S (1997) The epidemiology of hematogenous candidiasis caused by different Candida species. Clin Infect Dis 24: Akova M, Akalin HE, Uzun O, Gur D (1991) Emergence of Candida krusei infections after therapy of oropharyngeal candidiasis with fluconazole. Eur J Clin Microbiol Infect Dis 10: Arthington-Skaggs BA, Lee-Yang W, Ciblak MA, Frade JP, Brandt ME, Hajjeh RA, Harrison LH, Sofair AN, Warnock DW (2002) Comparison of visual and spectrophotometric methods of broth microdilution MIC end point determination and evaluation of a sterol quantitation method for in vitro susceptibility testing of fluconazole and itraconazole against trailing and nontrailing Candida isolates. Antimicrob Agents Chemother 46: Barchiesi F, Calabrese D, Sanglard D, Falconi DF, Caselli F, Giannini D, Giacometti A, Gavaudan S, Scalise G (2000) Experimental induction of fluconazole resistance in Candida tropicalis ATCC 750. Antimicrob Agents Chemother 44: Calvet HM, Yeaman MR, Filler SG (1997) Reversible fluconazole resistance in Candida albicans: a potential in vitro model. Antimicrob Agents Chemother 41: Chen YC, Chang SC, Sun CC, Yang LS, Hsieh WC, Luh KT (1997) Secular trends in the epidemiology of nosocomial fungal infections at a teaching hospital in Taiwan, 1981 to Infect Control Hosp Epidemiol 18:

6 180 Y.-L. Yang et al. / Diagnostic Microbiology and Infectious Disease 61 (2008) Cheng MF, Yu KW, Tang RB, Fan YH, Yang YL, Hsieh KS, Ho M, Lo HJ (2004) Distribution and antifungal susceptibility of Candida species causing candidemia from 1996 to Diagn Microbiol Infect Dis 48: Chou HH, Lo HJ, Chen KW, Liao MH, Li SY (2007) Multilocus sequence typing of Candida tropicalis shows clonal cluster enriched in isolates with resistance or trailing growth of fluconazole. Diagn Microbiol Infect Dis 58: Clinical and Laboratory Standards Institute (formally NCCLS) (1997) Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard. M27A. Wayne, PA: CLSI. Fleck R, Dietz A, Hof H (2007) In vitro susceptibility of Candida species to five antifungal agents in a German university hospital assessed by the reference broth microdilution method and Etest. J Antimicrob Chemother 59: Hadfield TL, Smith MB, Winn RE, Rinaldi MG, Guerra C (1987) Mycoses caused by Candida lusitaniae. Rev Infect Dis 9: Hung CC, Chen YC, Chang SC, Luh KT, Hsieh WC (1996) Nosocomial candidemia in a university hospital in Taiwan. J Formos Med Assoc 95: Hung CC, Yang YL, Lauderdale TL, McDonald LC, Hsiao CF, Cheng HH, Ho YA, Lo HJ (2005) Colonization of human immunodeficiency virusinfected outpatients in Taiwan with Candida species. J Clin Microbiol 43: Kontoyiannis DP (2002) Why prior fluconazole use is associated with an increased risk of invasive mold infections in immunosuppressed hosts: an alternative hypothesis. Clin Infect Dis 34: Leaw SN, Chang HC, Sun HF, Barton R, Bouchara JP, Chang TC (2006) Identification of medically important yeast species by sequence analysis of the internal transcribed spacer regions. J Clin Microbiol 44: Leaw SN, Chang HC, Barton R, Bouchara JP, Chang TC (2007) Identification of medically important Candida and non-candida yeast species by an oligonucleotide array. J Clin Microbiol 45: Lee MK, Williams LE, Warnock DW, Arthington-Skaggs BA (2004) Drug resistance genes and trailing growth in Candida albicans isolates. J Antimicrob Chemother 53: Lo HJ, Ho AH, Ho M (2001) Factors accounting for mis-identification of Candida species. J Microbiol Immunol Infect 34: Lo HJ, Cheng HH, TSARY Hospitals (2005) Distribution of clinical yeasts in Taiwan. Fung Sci 20: Marr KA, Lyons CN, Ha K, Rustad TR, White TC (2001) Inducible azole resistance associated with a heterogeneous phenotype in Candida albicans. Antimicrob Agents Chemother 45: Orozco AS, Higginbotham LM, Hitchcock CA, Parkinson T, Falconer D, Ibrahim AS, Ghannoum MA, Filler SG (1998) Mechanism of fluconazole resistance in Candida krusei. Antimicrob Agents Chemother 42: Peman J, Canton E, Gobernado M (2005) Epidemiology and antifungal susceptibility of Candida species isolated from blood: results of a 2-year multicentre study in Spain. Eur J Clin Microbiol Infect Dis 24: Pfaller MA, Messer SA, Hollis RJ, Jones RN, Doern GV, Brandt ME, Hajjeh RA (1999) Trends in species distribution and susceptibility to fluconazole among blood stream isolates of Candida species in the United States. Diagn Microbiol Infect Dis 33: Pfaller MA, Jones RN, Doern GV, Sader HS, Messer SA, Houston A, Coffman S, Hollis RJ (2000) Bloodstream infections due to Candida species: SENTRY antimicrobial surveillance program in North America and Latin America, Antimicrob Agents Chemother 44: Pfaller MA, Diekema DJ, Messer SA, Boyken L, Hollis RJ (2003) Activities of fluconazole and voriconazole against 1,586 recent clinical isolates of Candida species determined by Broth microdilution, disk diffusion, and Etest methods: report from the ARTEMIS Global Antifungal Susceptibility Program, J Clin Microbiol 41: Pfaller MA, Messer SA, Boyken L, Hollis RJ, Rice C, Tendolkar S, Diekema DJ (2004) In vitro activities of voriconazole, posaconazole, and fluconazole against 4,169 clinical isolates of Candida spp. and Cryptococcus neoformans collected during 2001 and 2002 in the ARTEMIS global antifungal surveillance program. Diagn Microbiol Infect Dis 48: Pfaller MA, Boyken L, Hollis RJ, Kroeger J, Messer SA, Tendolkar S, Diekema DJ (2007a) In vitro susceptibility of invasive isolates of Candida spp. to anidulafungin, caspofungin, and micafungin: six years of global surveillance. J Clin Microbiol 46: Pfaller MA, Diekema DJ, Gibbs DL, Newell VA, Meis JF, Gould IM, Fu W, Colombo AL, Rodriguez-Noriega E (2007b) Results from the ARTEMIS DISK Global Antifungal Surveillance study, 1997 to 2005: an 8.5-year analysis of susceptibilities of Candida species and other yeast species to fluconazole and voriconazole determined by CLSI standardized disk diffusion testing. J Clin Microbiol 45: Prasad KN, Agarwal J, Dixit AK, Tiwari DP, Dhole TN, Ayyagari A (1999) Role of yeasts as nosocomial pathogens and their susceptibility to fluconazole and amphotericin B. Indian J Med Res 110: Slavin MA, Osborne B, Adams R, Levenstein MJ, Schoch HG, Feldman AR, Meyers JD, Bowden RA (1995) Efficacy and safety of fluconazole prophylaxis for fungal infections after marrow transplantation a prospective, randomized, double-blind study. J Infect Dis 171: St Germain G, Laverdiere M, Pelletier R, Bourgault AM, Libman M, Lemieux C, Noel G (2001) Prevalence and antifungal susceptibility of 442 Candida isolates from blood and other normally sterile sites: results of a 2-year (1996 to 1998) multicenter surveillance study in Quebec, Canada. J Clin Microbiol 39: Walsh TJ, Hiemenz JW, Seibel NL, Perfect JR, Horwith G, Lee L, Silber JL, DiNubile MJ, Reboli A, Bow E, Lister J, Anaissie EJ (1998) Amphotericin B lipid complex for invasive fungal infections: analysis of safety and efficacy in 556 cases. Clin Infect Dis 26: Walsh TJ, Groll A, Hiemenz J, Fleming R, Roilides E, Anaissie E (2004) Infections due to emerging and uncommon medically important fungal pathogens. Clin Microbiol Infect 10(Suppl 1): Wang JS, Li SY, Yang YL, Chou HH, Lo HJ (2007) Association between fluconazole susceptibility and genetic relatedness among Candida tropicalis isolates in Taiwan. J Med Microbiol 56: White TC, Marr KA, Bowden RA (1998) Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 11: Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB (2004) Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis 39: Yang YL, Lo HJ (2001) Mechanisms of antifungal agent resistance. J Microbiol Immunol Infect 34: Yang YL, Cheng HH, Lo HJ (2004a) In vitro activity of voriconazole against Candida species isolated in Taiwan. Int J Antimicrob Agents 24: Yang YL, Ho YA, Cheng HH, Ho M, Lo HJ (2004b) Susceptibilities of Candida species to amphotericin B and fluconazole: the emergence of fluconazole resistance in Candida tropicalis. Infect Control Hosp Epidemiol 25: Yang YL, Ho YA, Cheng HH, Lo HJ (2005a) Distribution and susceptibility to amphotericin B and fluconazole of Candida spp. isolated from Taiwan. Epidemiol Infect 133: Yang YL, Li SY, Cheng HH, Lo HJ (2005b) Susceptibilities to amphotericin B and fluconazole of Candida species in TSARY Diagn Microbiol Infect Dis 51: Yang YL, Cheng HH, Lo HJ (2006) Distribution and antifungal susceptibility of Candida species isolated from different age populations in Taiwan. Med Mycol 44: